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Microbial Cell Factories
Open Access
Research
High yield recombinant thermostable -amylase production using
an improved Bacillus licheniformis system
Dandan Niu
1,3
, Zhirui Zuo
1
, Gui-Yang Shi
1,2
and Zheng-Xiang Wang*
1,2
Address:
1
Center for Bioresource and Bioenergy, School of Biotechnology & Culture and Information Center of Industrial Microorganisms of
China Universities, Jiangnan University, Wuxi 214122, PR China,
2
The Key Laboratory of Industrial Biotechnology, Ministry of Education, Wuxi
214122, PR China and
3
Institute for Wine Biotechnology, University of Stellenbosch, Matieland 7602, South Africa
Email: Dandan Niu - ndd2002@126.com; Zhirui Zuo - lygzuoz@sohu.com; Gui-Yang Shi - gyshi@jiangnan.edu.cn; Zheng-
Xiang Wang* - zxwang@jiangnan.edu.cn
* Corresponding author
Abstract
Background: Some strains of Bacillus licheniformis have been improved by target-directed
screening as well as by classical genetic manipulation and used in commercial thermostable -
amylase and alkaline protease production for over 40 years. Further improvements in production
of these enzymes are desirable.
Results: A new strain of B. licheniformis CBBD302 carrying a recombinant plasmid pHY-amyL for
Bacillus licheniformis -amylase (BLA) production was constructed. The combination of target-
directed screening and genetic recombination led to an approximately 26-fold improvement of BLA
production and export in B. licheniformis. Furthermore, a low-cost fermentation medium containing
soybean meal and cottonseed meal for BLA production in shake-flasks and in a 15 liter bioreactor
was developed and a BLA concentration of up to 17.6 mg per ml growth medium was attained.
Conclusion: This production level of BLA by B. licheniformis CBBD302(pHY-amyL) is amongst the
highest levels in Gram-positive bacteria reported so far.
Background
In any industrial biotechnological process, achieving high
productivity is an essential factor for commercial success.
The maximum specific productivities of a production
strain are usually independent of process parameters and
determined by the genetic and physiological properties of
the organism. Bacterial extracellular enzymes are an
important class of industrial enzymes constituting
approximately 20% of the enzyme market [1,2]. To obtain
a high yield in bacterial extracellular enzyme production,
the following genetic and physiological properties of the
strain are important: a) the metabolic flux for amino acids
synthesis and ATP regeneration, b) the cell growth rate
and cell density in an inexpensive medium, c) mainly veg-
etative growth by spore-forming strains, d) secretion
capacity for extracellular enzymes, e) long-term preserva-
tion in an active form in broth, and f) a high expression
level of the specific gene encoding a bacterial extracellular
enzyme [3].
Genetic improvement of bacterial extracellular enzyme
production is achieved by applying a range of strategies
based on molecular cloning tools. These include: 1)
enhancement of expression level through amplification of
gene copy number [4], codon usage optimization [5], or
strong promoters being used to boost gene transcription
Published: 31 October 2009
Microbial Cell Factories 2009, 8:58 doi:10.1186/1475-2859-8-58
Received: 31 March 2009
Accepted: 31 October 2009
This article is available from: http://www.microbialcellfactories.com/content/8/1/58
2009 Niu et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Microbial Cell Factories 2009, 8:58 http://www.microbialcellfactories.com/content/8/1/58
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(page number not for citation purposes)
[6]; 2) enhancement of secretion by modulation of signal
peptides [7,8], fusion to heterologous signal peptides for
efficient targeting to the translocase [9], increasing the
copy number of signal peptidase genes [10,11], or dereg-
ulation and/or co-expression of chaperon encoding genes
to make efficient protein folding [12]; 3) improvement of
productivity through re-designing the capacity of the
secretion machinery by targeted deletion of genes encod-
ing non-beneficial extracellular enzymes [13], or genome
reduction [14]; and 4) improvement of the productivity
by preventing degradation of extracellular enzymes using
protease deficient strains [15].
In general, a specific strain should possess a definite max-
imum capacity for synthesis and secretion of extracellular
enzymes [4]. Overproduction of secreted proteins some-
times severely affects the secretory system of an organism
[16] and eventually results in a secretion stress response
that may limit secretion [17,18]. Moreover, it is almost
impossible at the present stage to enhance the maximum
synthesis and secretion capacity by site-directed gene dis-
ruption and expression in a specific strain. Alternatively,
data from genome shuffling and genome size reduction
studies [14] strongly suggest that a natural strain should
exist with a maximum capacity for synthesis and secretion
of extracellular enzymes as a result of accumulation of
mutations and shuffling of the genomes.
In present work, the combination of target-directed
screening and genetic recombination yielded a novel B.
licheniformis strain that produced up to 17.6 mg B. licheni-
formis -amylase per ml growth medium.
Results and discussion
Rationale for the selection approach of the B.
licheniformis strain and property identification
Enzyme synthesis and export is an energy-dependent
event [3]. In order to select a suitable host cell for BLA
overproduction, following genetic and physiological crite-
ria were applied: 1) the ability of the strain to sporulate
should be poor in order to extend the duration of BLA
production; 2) the strain should form little or no
lichenysin in order to reduce consumption of ATP and the
amino acid pool; 3) the strain should not clump during
cultivation to maintain efficiency in a production bioreac-
tor; 4) the strain should grow well on either low-cost fer-
mentation medium containing a high substrate
concentration appropriate for an industrial process; 5) the
strain should contain no native plasmids but be sensitive
to kanamycin or tetracyclin to facilitate further genetic
manipulation and 6) the strain should be amenable to
transformation to enable genetic modification.
B. licheniformis strain selection and biological property
identification
Following a selection procedure described above, based
on these criteria, a candidate strain designated as
CBB0302 was selected out of a total of 526 B. licheniformis
isolates (Figure 1). The strain produced catalase, amylase
and protease, utilized citrate, propionate and nitrate, grew
in 7% NaCl, and at 50C but not at 60C, typical of B.
licheniformis strains. Notably, fewer than 0.5% of the cells
in culture formed spores and no cell clumping was found
after 72 h cultivation. Furthermore the strain did not pro-
duce lichenysin-like pigments and harboured no native
plasmids. The cell shape of the strain CBB0302 was iden-
tical to that of B. licheniformis CICIM B30306, an indus-
trial BLA-producing strain, when both strains were
cultivated in LB medium for 10-12 h at 45C and 220 rpm
but the average cell volume was 30-40% less (Figure 2).
Increased transformation efficiency by genetic
modification
The transformation efficiency of the natural B. licheni-
formis cells is poor and strains routinely need long periods
with difficulty to finally obtain a desired transformant
[[19]; our own unpublished results]. This is mainly due to
the existence of two type I restriction modification sys-
tems (RMS) in B. licheniformis [20,21]. Single as well as
double knock-outs of the RMS resulted in strains being
readily transformable with plasmids isolated from Bacilli.
Introduction of shuttle plasmids isolated from Escherichia
coli is routinely possible when the double mutant B.
licheniformis MW3 (hsdR1, hsdR2) was used in transfor-
mation experiments [19].
A strain CBBD302 was developed by deletion of a type I
RMS locus in strain CBB0302 by using homolog-medi-
ated recombination according to the method described by
Waschkau et al [19]. The growth and secretion of extracel-
lular enzymes by this strain were unaffected by the dele-
tion (data not shown). The transformation efficiency with
a shuttle plasmid pHY-300PLK isolated from E. coli was
significantly improved and 42 cfu/g DNA in strain
CBBD302 but only 5 cfu/g DNA in strain CBB0302 were
attained.
Increased recombinant BLA production and secretion by
using B. licheniformis CBBD302 as host
In order to test BLA production in B. licheniformis
CBBD302, a recombinant plasmid pHY-amyL was con-
structed. A 1.6 kb fragment containing B. licheniformis
B0204 amyL coding for the mature BLA peptide was recov-
ered from pET28a-amyL
NEW
by PCR, inserted into the
EcoRI and SmaI sites of pHY-WZX and functionally tested
in E. coli (Figure 3), yielded hybrid plasmid pHY-amyL.
Subsequently, pHY-amyL was transferred into B. licheni-
formis CBBD302, yielding B. licheniformis CBBD302 (pHY-
Microbial Cell Factories 2009, 8:58 http://www.microbialcellfactories.com/content/8/1/58
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amyL). The BLA production was carried out in LB supple-
mented with 40 g/l lactose in shake-flasks (Figure 4). B.
licheniformis CBBD302, B. licheniformis B30306, B. licheni-
formis B0204 and B. licheniformis CBBD302 (pHY-amyL)
produced 0.1, 0.7, 0.9 and 2.6 mg BLA per ml growth
medium, respectively. Strain CBBD302 carrying pHY-
amyL produced a 26-fold improvement in BLA produc-
tion compared to the parent strain CBBD302 and about
three times compared to the B. licheniformis B0204.
Although the growth rate was slower than its parent
CBBD302, strain CBBD302 (pHY-amyL) grew signifi-
cantly faster than industrial strains B0204 and B30306 did
(Figure 4).
Bacterial extracellular enzyme production is a complex
process, in which the efficiencies of transcription and
translation of the enzyme-encoding genes as well as pro-
tein translocation define the enzyme concentration in the
growth medium and are under control of the bacterial
host. A B. licheniformis strain has been found to have a spe-
B. licheniformis strain selection strategy and selection Figure 1
B. licheniformis strain selection strategy and selection. A: selection routine; B: selection results.
Microbial Cell Factories 2009, 8:58 http://www.microbialcellfactories.com/content/8/1/58
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cific capacity maximum for protein synthesis and secre-
tion by the introduction of different copy numbers of
amyL in B. licheniformis B0204 [3]. Evidently the capacity
could be improved since B. licheniformis CBB0302 with
physiological properties of less than 0.5% sporulation, no
production of lichenysin-like pigments, reduced nutrient
requirement for growth, smaller cell volume, identical cell
shape and no cell clumping during propagation gave an
increased BLA yield.
Optimization of fermentation medium
-Amylase production is subjected to catabolite repres-
sion by glucose and other sugars, similar to most induci-
ble enzymes. Therefore, the use of glucose in the
production of -amylase in certain cases is problematic
[22,23]. A number of other substrates such as lactose [24]
have also been used for the production of -amylase. The
effect of the addition of different carbon sources to the fer-
mentation medium on BLA production was investigated
and the results are summarized in Figure 5. BLA secretion
by strain CBBD302 carrying pHY-amyL varied between
carbon sources and the lactose gave the highest BLA con-
centration and glucose, starch and corn cob hydrolysate
supported a 70~80% BLA production level compared to
lactose (Figure 5). These results indicate that many vari-
ous carbon sources can be used for BLA production and
BLA synthesis in CBBD302 is not subject to catabolite
repression.
Yeast extract alone or in conjunction with other nitrogen
sources such as bactopeptone and ammonium sulfate has
been used for the production of -amylase from Bacillus
sp. [25]. In this study, the effect of various nitrogen
sources in the fermentation medium on BLA production
was investigated. When soybean meal, fish meal or
(NH
4
)
2
SO
4
was added as nitrogen source for BLA produc-
tion to the fermentation medium, lower respective BLA
production levels of 95.8, 28.6 and 25.3% were obtained
relative to cottonseed meal. Much higher BLA production
levels were obtained with cottonseed meal and/or soy-
bean meal supplemented 0.01 mol/l ammonium sul-
phate as nitrogen source (respectively 132 and 140% of
cottonseed level).
The maximum production of BLA by B. licheniformis
CBBD302(pHY-amyL) of 17.6 mg/ml was obtained when
the strain was cultivated in the fermentation medium con-
sisting of 40 g/l lactose, 25 g/l soybean meal, 20 g/l cot-
tonseed meal, 30 g/l corn-steep liquor and 0.01 mol/l
ammonium sulfate in a pH-controlled 15 l bioreactor fer-
mentations (Figure 6). The BLA production was boosted
by the 40 g/l lactose when fed at the 54
th
hour.
Conclusion
The combination of target-directed screening and genetic
recombination led to an overall 26-fold improvement of
BLA production and export in B. licheniformis. In a low-
cost fermentation medium containing 40 g/l lactose, 25 g/
l soybean meal, 20 g/l cottonseed meal, 30 g/l corn-steep
liquor and 0.01 mol/l ammonium sulphate and a 15 l
bioreactor up to 17.6 mg BLA per ml growth medium was
produced. This production level of BLA by B. licheniformis
CBBD302(pHY-amyL) is amongst the highest levels in
Gram-positive bacteria reported so far.
The scanning electron micrograph of B. licheniformis Figure 2
The scanning electron micrograph of B. licheniformis. 10
8
cells were inoculated in a 250 ml flask with 30 ml of LB
medium and cultivated for 10-12 h at 45C and 220 rpm. A drop of culture was collected and analyzed by scanning electron
microscope. A: B. licheniformis CICIM B30306; B: B. licheniformis CBB0302.
Microbial Cell Factories 2009, 8:58 http://www.microbialcellfactories.com/content/8/1/58
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Methods
Plasmids and strains
Plasmid pHY300PLK [26] was used to determine transfor-
mation efficiency and plasmid pHY-WZX [27] was used as
an expression vector in B. licheniformis. E. coli XL1 was
used as a host cell for functional identification of the
recombinant plasmid. Molecular biology methods were
described previously [28]. A 1.6 kb fragment containing
amyL coding for the mature peptide as well as its 180 bp
downstream sequence was amplified with primers F2-
EcoRI (5'-CGGAATTCCTTAATGGGACGCTGATGC-3')
and R1-SmaI (5'-TACCCGGGTACATCAGATAACGTT-
GCC-3') using pET28a-amyL
NEW
[29] as template. The
amplified product was purified and digested with EcoRI
and SmaI and subsequently cloned into the same sites of
pHY-WZX to yield recombinant pHY-amyL. B. licheni-
formis isolates as well as B0204 and B030306 were pur-
chased from CICIM-CU http://cicim-cu.jiangnan.edu.cn.
CBBD302 was developed from a native B. licheniformis
isolate CBB0302, in which the restriction modification
system locus was deleted according to the method
described by Waschkau et al [19]. B. licheniformis
CBBD302(pHY-amyL) was CBBD302 harboring pHY-
amyL by using electroporation [30].
Strain screening and biological property identification
B. licheniformis strains were recovered from a culture col-
lection held at -70C and single colonies were picked
Development and identification of pHY-amyL Figure 3
Development and identification of pHY-amyL. A: The
physical map of pHY-amyL. B: -Amylase was expressed by
E. coli carrying pHY-amyL, a: E. coli(pHY-WZX); b: E.
coli(pHY-amyL).
BLA production by B. licheniformis CBBD302(pHY-amyL) Figure 4
BLA production by B. licheniformis CBBD302(pHY-
amyL). B. licheniformis CBBD302 (circles), B. licheniformis
CBBD302(pHY-amyL) (triangles), B. licheniformis B30306
(squares) and B. licheniformis B0204 (diamonds) were culti-
vated in LB medium supplemented with 40 g/l lactose for up
to 120 h at 42C and 220 rpm. Open symbols for cell growth
and closed symbols for BLA production.
O
5
1O
15
2O
12 24 4O 56 72 88 1O4 12O
Tine (h)
C
e
I
I

D
e
n
s
i
l
y

(
O
D
6
O
O
n
n
)
O.O
O.4
O.8
1.2
1.6
2.O
2.4
2.8

L
A

(
n
g
/
n
I
)
The time-course for BLA production in B. licheniformis CBBD302 (pHY-amyL) on different carbon sources Figure 5
The time-course for BLA production in B. licheni-
formis CBBD302 (pHY-amyL) on different carbon
sources. Symbols: triangle, lactose; square, glucose; dia-
mond, starch; circle, corn cob hydrolysate. The highest activ-
ity of BLA on lactose is designated as 100% activity.
0
10
20
30
40
50
60
70
80
90
100
24 36 48 60 72 84 96 108 120
Time (h)
B
L
A

a
c
t
i
v
i
t
y

(
%
)
Microbial Cell Factories 2009, 8:58 http://www.microbialcellfactories.com/content/8/1/58
Page 6 of 7
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onto LB plate and cultivated at 45C for up to 72 h. Col-
onies with no or reduced lichenysin formation were
picked onto LB plate supplemented with 5 g/ml kanamy-
cin or 10 g/ml tetracycline and cultivated for 48 h. Colo-
nies that failed to grow were examined for the existence of
the native plasmids by plasmid extraction and agarose
electrophoresis as described previously [28]. Strains carry-
ing no native plasmids and sensitive to kanamycin and
tetracycline were cultivated in LB containing 2% glucose
at 45C for up to 72 h and their degree of sporulation and
cells dispersion were checked by light microscopy. Strains
with sporaulation rate less than 5% after 72 h and with no
cell clumping were inoculated onto LB supplemented
30% glucose or 30% starch and cultivated at 45C for 24
h. The rapidly growing strains were selected and cultivated
at 45C and 24 h on a nutrient limited medium consist-
ing of 0.02% peptone, 0.01% yeast extract, 1% NaCl and
0.01% glucose. Those strains that grew well on the nutri-
ent limited medium were examined for their biological
properties including the major secreted enzymes, genetic
transformation efficiency as well as plasmid stability as
described by Zhuge & Wang [31]. The cells were examined
with a Quanta-200 scanning electron microscope (FEI,
Netherland).
Cultivation
E. coli XL1 was cultivated at 37C in LB medium. As
required, 100 g/ml ampicillin and/or 25 g/ml kanamy-
cin were added to the medium. B. licheniformis B0204,
B030306 and CBBD302 were cultivated at 42C in LB
medium. For the shake-flask fermentation evaluation, B.
licheniformis strains were grown in 500 ml Erlenmeyer
flasks containing 50 ml LB supplemented with 40 g/l lac-
tose at 42C and 220 rpm. For optimization studies the
fermentation medium consisted of 30 g/l corn-steep liq-
uor, 30 g/l nitrogen source (cottonseed meal, soybean
meal and fish meal, and former two combination with
0.01 mol/l (NH
4
)
2
SO
4
and 40 g/l) carbon source (lactose,
glucose, starch or corn cob hydrolysate) at pH 6.0 was
used. For bioreactor cultivation studies, a Biostat (B.
Braun, Melsungen, Germany) with a 15 l working volume
was used. The bioreactor was inoculated with 5% (v/v)
broth and cultivated at 42C with a controlled pH of 6.0.
Analytical procedures
For shake flask cultivation, samples for cell density and
BLA activity were taken at regular intervals. The optical
density (OD
600 nm
) was measured in triplicate with an
Ultrospec 3100 pro spectrophotometer (Amersham Phar-
macia, UK). SDS-PAGE was performed using a Mini Pro-
tean 3 apparatus (Bio-Rad, USA). Proteins were stained by
Coomassie Brilliant Blue G250. Directly after sampling,
BLA activity was measured spectrophotometrically (Ultro-
spec 3100 pro, Amersham Pharmacia, UK) as described
previously [32]. One unit was defined as the amount of
enzyme that hydrolyzes 1 mg water soluble corn starch
per minute at 70C and pH 6.0. The amount of BLA pro-
tein (g/ml) in the medium was calculated using BLA spe-
cific activity parameter of 1 mg BLA is equal to 996 U.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
DN carried out the molecular genetic studies. ZZ partici-
pated in the design of the study and performed the statis-
tical analysis. G-YS participated fermentation
experiments. Z-XW conceived of the study and partici-
pated in its design and coordination. All authors read and
approved the final manuscript.
Acknowledgements
This work was financially supported by China High-Tech Research and
Development Program 2006AA020204 and partly granted by Sino-South
Africa Cooperation Program 2009DFA31300 and NRF67185. The authors
thankfully thank Dr. B.A. Prior from University of Stellenbosch for the man-
uscript preparation and language revision.
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L
A

(
m
g
/
m
l
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